Microfluidic devices incorporating improved channel geometries

ABSTRACT

The present invention generally provides microfluidic devices which incorporate improved channel and reservoir geometries, as well as methods of using these devices in the analysis, preparation, or other manipulation of fluid borne materials, to achieve higher throughputs of such materials through these devices, with lower cost, material and/or space requirements.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S.application Ser. No. 09/372,655 now U.S. Pat. No. 6,153,073 filed Aug.11, 1999, which is a continuation of U.S. application Ser. No.09/165,704 now U.S. Pat. No. 6,235,175 filed Oct. 2, 1998, thedisclosure of which is incorporated by reference for all purposes.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/845,754, now U.S. Pat. No. 5,976,336 filed Apr. 25, 1997.This application also claims priority to Provisional U.S. Patentapplication No. 60/060,902, filed Oct. 3, 1997. Each of the abovereferenced applications is hereby incorporated herein by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

There has been a growing interest in the development and manufacturingof microscale fluid systems for the acquisition of chemical andbiochemical information, in both preparative and analytical capacities.Adaptation of technologies from the electronics industry, such asphotolithography, wet chemical etching and the like, to these fluidicsystems has helped to fuel this growing interest.

One of the first areas in which microscale fluid systems have been usedfor chemical or biochemical analysis has been in the area of capillaryelectrophoresis (CE). CE systems generally employ fused silicacapillaries, or more recently, etched channels in planar silicasubstrates, filled with an appropriate separation matrix or medium. Asample fluid that is to be analyzed is injected at one end of thecapillary or channel. Application of a voltage across the capillary thenpermits the electrophoretic migration of the species within the sample.Differential electrophoretic mobilities of the constituent elements of asample fluid, e.g., due to their differential net charge or size,permits their separation, identification and analysis. For a generaldiscussion of CE methods, see, e.g., U.S. Pat. No. 5,015,350, toWiktorowicz, and U.S. Pat. No. 5,192,405 to Petersen et al.

Fabrication of CE systems using planar chip technology has also beendiscussed. See, e.g., Mathies et al., Proc. Nat'l Acad. Sci. (1994)91:11348–11352, Jacobsen et al., Anal. Chem. (1994) 66:1114–1118,Effenhauser et al., Anal. Chem. (1994) 66: 2949–2953. However,typically, such systems employ a single sample introduction point, e.g.,a single well for introducing samples that are to be analyzed in thecapillary channel. This requires rinsing and reloading the well prior toeach analysis. Further, where one wishes to analyze larger numbers ofsamples, larger components of each sample, e.g., large nucleic acidfragments, proteins and the like, can build up within the sample loadingand separation channels, and/or adsorb to capillary walls, eventuallyaffecting the operation of the system.

It would therefore be desirable to provide microfluidic devices,including CE systems, which permit faster analysis of multiple samples,and do so with minimal and even reduced cost, space and timerequirements. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a microfluidic devicethat comprises a planar substrate having a first surface. At least thefirst, second and third microscale channels are disposed in the interiorportion, the second channel intersecting the first channel at a firstintersection, and the third channel intersecting the first channel at asecond intersection. A plurality of sample reservoirs is disposed in thebody structure with each of the sample reservoirs being connected to thesecond channel. At least a first waste reservoir is connected to thethird channel.

The present invention also provides a microfluidic device as describedabove, with at least one sample reservoir being connected to the secondchannel and at least one sample reservoir being connected to the thirdchannel. The device also includes at least first and second wastereservoirs, the first waste reservoir being connected to the firstchannel, and the second waste reservoir being connected to the secondchannel.

In another aspect, the present invention also provides a microfluidicdevice as described above, but comprising a preloading module incommunication with the first channel. The preloading module comprises afirst sample loading channel intersecting the first channel at a firstintersection. The preloading module also includes a first plurality ofsample reservoirs in fluid communication with the first sample loadingchannel and a first load/waste reservoir in communication with the firstsample loading channel between the first plurality of sample reservoirsand the first intersection.

This invention also provides a method of analyzing a plurality ofsamples using the microfluidic device as described above. A plurality ofsample reservoirs is disposed in the body structure, each of the samplereservoirs being connected to the second channel. At least a first wastereservoir is connected to the third channel. The method also involvestransporting a sample material from the first of the plurality of samplereservoirs through the second channel, through the first and secondintersections, into the third channel, toward the first waste reservoir.A portion of the sample material is injected at the first intersectioninto the first channel, transported along the first channel, andanalyzed in the analysis channel.

In a related aspect, the present invention provides a method ofseparating component elements of a sample material, using themicrofluidic device described above, but comprising a plurality ofsample reservoirs. Each of the sample reservoirs is connected to thesecond channel and at least a first waste reservoir is connected to thethird channel. The method also involves transporting the sample materialfrom a first of said plurality of sample reservoirs through the secondchannel, through the first and second intersections, into the thirdchannel, toward the first waste reservoir. A portion of the samplematerial is injected at the first intersection into the first channeland transported along the first channel to separate the componentelements of the sample material.

The present invention also provides for the use of a microfluidic devicethat includes a body structure having an interior portion and anexterior portion, at least first, second and third microscale channelsdisposed in the interior portion, the second channel intersecting thefirst channel at a first intersection, the third channel intersectingthe first channel at a second intersection, a plurality of samplereservoirs in communication with the second channel having a pluralityof different sample materials disposed therein, and a waste reservoir incommunication with the third channel, in separating component elementsof the sample materials.

Another aspect of the invention provides a microfluidic devicecomprising an analysis channel and a sample loading channel in fluidcommunication with the analysis channel at a first intersection. Aplurality of sample sources is in fluid communication with the sampleloading channel, whereby there is at least one of the plurality ofsample sources in fluid communication with the sample loading channel oneach side of the first intersection. First and second load/wastechannels intersect the sample loading channel at second and thirdintersections, respectively. The second and third intersections are ondifferent sides of the first intersection.

In a further aspect, the present invention provides a microfluidicdevice comprising an analysis channel. A sample loading channel is on afirst side of said analysis channel, and intersects the analysis channelat a first intersection. A plurality of sample reservoirs is in fluidcommunication with the sample loading channel on a first side of thefirst intersection and a waste channel is on a second side of theanalysis channel, intersecting the analysis channel at a secondintersection. A waste reservoir is in fluid communication with the wastechannel on the second side of the first intersection.

Another aspect of the invention provides a microfluidic devicecomprising an analysis channel. A sample loading channel intersects theanalysis channel at a first intersection. The device also includes asample preloading module which comprises a plurality of samplereservoirs and a waste reservoir disposed in the body structure. Each ofthe plurality of sample reservoirs and waste reservoir is in fluidcommunication with the sample loading channel on the same side of thefirst intersection.

In an additional aspect, the present invention provides a microfluidicdevice comprising an analysis channel, and also including first andsecond transverse channels disposed in the interior portion. The firsttransverse channel is located on a first side of the analysis channel,intersecting the analysis channel at a first intersection. The secondtransverse channel is located on a second side of the analysis channel,intersecting the analysis channel at a second intersection. A firstsample source is placed in fluid communication with the first transversechannel, and a second sample source is placed in fluid communicationwith the second transverse channel. A first waste channel is located atthe first transverse channel at a third intersection and a second wastechannel is located at the second transverse channel at a fourthintersection. The device also contains a material direction system forindividually transporting a sample from each of the first and secondsample sources to the first and second waste channels via the first andsecond transverse channels, respectively, and selectively injecting thesamples into the analysis channel.

In yet another aspect, the present invention provides a microfluidicdevice comprising an analysis channel, and first and second transversechannels disposed in said interior portion. The first transverse channelis disposed on a first side of the analysis channel, intersecting theanalysis channel at a first intersection. The second transverse channelis disposed on a second side of the analysis channel, intersecting theanalysis channel at a second intersection. A plurality of sample sourcesis in fluid communication with the first transverse channel. A firstwaste channel is disposed in the interior portion and intersects thefirst transverse channel at a third intersection. At least a secondwaste channel is disposed in the interior portion and intersects thesecond transverse channel at a fourth intersection. The device alsoincludes a material direction system for individually transporting asample from each of the first and second sample sources to the first andsecond waste channels via the first and second transverse channels,respectively, and selectively injecting the samples into the analysischannel.

Another aspect of the invention provides a microfluidic devicecomprising an analysis channel and a sample loading channel disposed influid communication with the analysis channel. A plurality of samplesources is also provided in fluid communication with the sample loadingchannel.

In still a further aspect, the invention provides a method for analyzinga plurality of different materials with a microfluidic device whichincludes an analysis channel. A sample loading channel is disposed indevice and intersects said analysis channel at a first intersection. Aplurality of sample sources is in fluid communication with said sampleloading channel. A first sample is transported from a first of saidplurality of sample sources, through said sample loading channel to saidfirst intersection. A portion of said first sample is injected into saidanalysis channel; analyzing said portion of said first sample in saidanalysis channel. A second sample is transported from a second of saidplurality of sample sources through said loading channel to saidintersection. A portion of said second sample is injected into saidanalysis channel; analyzing said portion of said second sample in saidanalysis channel.

A further aspect of the invention provides a method of performinganalysis on a plurality of different sample materials with amicrofluidic device which comprises a planar substrate having a firstsurface with an analysis channel. The device also includes a sampleloading channel which intersects said analysis channel at a firstintersection, a sample preloading module which comprises at least firstand second sample reservoirs and a waste reservoir disposed in said bodystructure, wherein each of said plurality of sample reservoirs and saidwaste reservoir are in fluid communication with said sample loadingchannel. A sample is transported from said first sample reservoir tosaid first intersection. A portion of said first sample is injected intosaid analysis channel. Said portion of said first sample is concurrentlyanalyzed in said analysis channel, and a second sample from said secondsample reservoir is transported into said loading channel and then tosaid waste reservoir. Said second sample is transported from saidloading channel to said intersection and injected into said analysischannel and analyzed in said analysis channel.

The present invention also provides a microfluidic device that includesa body structure with an analysis channel disposed therein. A pluralityof sample sources is also disposed in the body structure, where eachsample source is in fluid communication with a first point in theanalysis channel via one or more sample channels. The channel distancebetween a first of the plurality of sample sources and the point in theanalysis channel, is substantially equal to a channel distance between asecond of the plurality of sample sources and the point in the analysischannel.

It is a further aspect of the invention to provide a microfluidic devicecomprising a body structure with an analysis channel and a first sampleintroduction channel disposed in the body structure, where the sampleintroduction channel intersects the analysis channel at a first point. Afirst plurality of sample sources is disposed in the body structure,where each of the first plurality of sample sources is in fluidcommunication with the first sample introduction channel via a firstplurality of separate sample channels disposed in the body structure,respectively. The channel distance between a first of the firstplurality of sample sources and the first point is substantially equalto a channel distance between a second of the plurality of samplesources and the first point.

Another aspect of the invention is a microfluidic device comprising abody structure with an analysis channel and a sample loading channelintersecting and in fluid communication with the analysis channel. Inaccordance with this aspect of the invention, the analysis channel andthe sample loading channels typically have a width of less than 50 μm. Aplurality of sample sources is also provided in fluid communication withsaid sample loading channel.

In a related aspect, the present invention provides a method ofmanufacturing a microfluidic device comprising fabricating a pluralityof channels in a first planar surface of a first substrate. Theplurality of channels typically includes an analysis channel, a sampleloading channel disposed on a first side of the analysis channel andintersecting the analysis channel at a first intersection. Also includedis a plurality of sample channels intersecting the sample loadingchannel on a first side of the first intersection, and a waste channeldisposed on a second side of the analysis channel, and intersecting theanalysis channel at a second intersection. A second planar substrate isoverlaid on the planar surface of the first substrate to seal theplurality of channels. The second planar substrate has a plurality ofports disposed therethrough, which is comprised of two ports incommunication with opposite ends of the analysis channel, a waste portin communication with an unintersected terminus of the waste channel,and a plurality of sample ports each in separate communication with theunintersected termini of the sample channels.

A further aspect of the invention provides a microfluidic device havingan analysis channel and a sample loading channel which intersects theanalysis channel at a first intersection. Also included is a pluralityof sample sources disposed in fluid communication with the sampleloading channel, for analysis of each of the plurality of samples.

In an additional aspect, the present invention provides kits embodyingthe methods and apparatus herein. Kits of the invention optionallycomprise one or more of the following: (1) an apparatus or apparatuscomponent as described herein; (2) instructions for practicing themethods described herein, and/or for operating the apparatus orapparatus components herein; (3) one or more assay component; (4) acontainer for holding apparatus or assay components, and, (5) packagingmaterials.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–11 schematically illustrates the channel reservoir geometriesemployed in the devices of the present invention, and their operation inloading and injection of multiple samples FIGS. 1A through 1E and insample preloading (FIGS. 1F through 1I).

FIG. 2 is a schematic illustration of the chronology of the variousmaterial transport steps involved in performing capillaryelectrophoresis in a microfluidic device of the present invention(bottom) as compared to a CE system lacking a preloading feature (top).

FIG. 3 illustrates one embodiment of a microfluidic device incorporatingan improved channel/sample well geometry for performing serial analysisof multiple samples.

FIG. 4 illustrates another embodiment of a microfluidic deviceincorporating an improved channel/sample well geometry for performingserial analysis of multiple samples.

FIG. 5 illustrates still another alternate channel geometry in amicrofluidic device for performing serial analysis of multiple samples.

FIG. 6 is a plot of retention times for fluorescently dyed nucleic acidfragments injected into a CE channel fabricated into a substrateemploying the improved channel/sample well geometry of the presentinvention.

FIGS. 7A–7C are plots of fluorescence vs. time for a set of PCRfragments intercalated with a fluorescent dye (FIG. 7A), PhiX174 DNA,cleaved with HaeIII and intercalated with a fluorescent dye (FIG. 7B)and a buffer blank (FIG. 7C), serially injected into the analysischannel of a microfluidic device incorporating the channel/sample wellgeometry of the present invention.

FIGS. 8A and 8B illustrate nucleic acid separation analyses performed inmicrofluidic devices having 30 μm channel widths (FIG. 8A) and 70 μmchannel widths (FIG. 8B).

DETAILED DESCRIPTION OF THE INVENTION

I. General

The present invention generally provides microfluidic devices whichincorporate improved channel and reservoir geometries, as well asmethods of using these devices in the analysis, preparation, or othermanipulation of fluid borne materials, to achieve higher throughputs ofsuch materials through these devices, with lower cost, material and/orspace requirements.

As used herein, the term “microfluidic device or system” generallyrefers to a device or system which incorporates at least twointersecting channels or fluid conduits, where at least one of thechannels has at least one cross sectional dimension in the range of fromabout 0.1 to about 500 μm, preferably from about 1 to about 100 μm.

The microfluidic devices of the present invention comprise a centralbody structure in which the various microfluidic elements are disposed.The body structure includes an exterior portion or surface, as well asan interior portion which defines the various microscale channels and/orchambers of the overall microfluidic device. For example, the bodystructures of the microfluidic devices of the present inventiontypically employ a solid or semi-solid substrate that is typicallyplanar in structure, i.e., substantially flat or having at least oneflat surface. Suitable substrates may be fabricated from any one of avariety of materials, or combinations of materials. Often, the planarsubstrates are manufactured using solid substrates common in the fieldsof microfabrication, e.g., silica-based substrates, such as glass,quartz, silicon or polysilicon, as well as other known substrates, i.e.,gallium arsenide. In the case of these substrates, commonmicrofabrication techniques, such as photolithographic techniques, wetchemical etching, micromachining, i.e., drilling, milling and the like,may be readily applied in the fabrication of microfluidic devices andsubstrates. Alternatively, polymeric substrate materials may be used tofabricate the devices of the present invention, including, e.g.,polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA),polyurethane, polyvinylchloride (PVC), polystyrene polysulfone,polycarbonate, polymethylpentene, polypropylene, polyethylene,polyvinylidine fluoride, ABS (acrylonitrile-butadiene-styrenecopolymer), and the like. In the case of such polymeric materials,injection molding or embossing methods may be used to form thesubstrates having the channel and reservoir geometries as describedherein. In such cases, original molds may be fabricated using any of theabove described materials and methods.

The channels and chambers of the device are typically fabricated intoone surface of a planar substrate, as grooves, wells or depressions inthat surface. A second planar substrate, typically prepared from thesame or similar material, is overlaid and bonded to the first, therebydefining and sealing the channels and/or chambers of the device.Together, the upper surface of the first substrate, and the lower matedsurface of the upper substrate, define the interior portion of thedevice, i.e., defining the channels and chambers of the device.

In the devices described herein, at least one main channel, also termedan analysis channel, is disposed in the surface of the substrate throughwhich samples are transported and subjected to a particular analysis.Typically, a number of samples are serially transported from theirrespective sources, and injected into the main channel by placing thesample in a transverse channel that intersects the main channel. Thischannel is also termed a “sample loading channel.” The sample sourcesare preferably integrated into the device, e.g., as a plurality of wellsdisposed within the device and in fluid communication with the sampleloading channel, e.g., by an intermediate sample channel. However, thedevices of the invention may also include sample sources that areexternal to the body of the device, per se, but still in fluidcommunication with the sample loading channel.

The sample in the loading channel is drawn or transported across theintersection of the loading channel with the analysis channel. Thevolume or ‘plug’ of sample that is disposed within the intersection ofthese two channels is then drawn down the analysis channel whereupon itis subjected to the desired analysis. The intersection of two channels,e.g., as in the main channel and loading channel, may be a “T” or“three-way” intersection, where the loading channel intersects with andterminates in the main channel, or vice versa. Alternatively, the twochannels may intersect and cross each other, creating a “four-way”intersection. In this case, the volume of a sample that is injected isdirectly related to the volume of the intersection. Where larger volumesof samples are desired, one may generally stagger the intersection ofthe inlet side of the sample loading channel, e.g., the sample side, andthe intersection of the outlet side of the loading channel, e.g., thewaste side, whereby more sample is disposed within the analysis channelduring loading, e.g., as defined by the length of the analysis channelbetween the staggered intersections.

For ease of discussion, the devices and systems of the present inventionare generally described in terms of the performance of capillaryelectrophoretic analysis (CE) on a sample. Accordingly, for suchoperations, the main or analysis channel generally includes a sievingmatrix, buffer or medium disposed therein, to optimize theelectrophoretic separation of the constituent elements of the sample.However, it will be appreciated upon reading the instant disclosure thatthe microfluidic devices incorporating the improved geometries describedherein are also applicable to a wide variety of non-CE applications, andmay be used to perform any of a number of different analytical reactionson a sample, e.g., as described in commonly assigned InternationalApplication No. WO 98/00231, which is hereby incorporated herein byreference in its entirety for all purposes.

As noted above, the devices of the present invention employ channel andreservoir geometries that reduce the costs associated with producing thedevice, by reducing the amount of material required to fabricate thedevice itself. In addition, the devices of the invention are able toperform analyses with a much higher throughput rate, as well asfacilitate those analyses, by: (1) reducing the distance which aparticular sample must travel, or be transported, from its origin on thedevice to the analysis region or channel; (2) equalizing the distancethat any two samples must travel from their origins to the analysisregion or channel, and thus, equalize any effect that such transport hason that sample; (3) increasing the number of samples that may be placedinto a single device; (4) allowing one sample to be analyzed whileanother is being drawn into place, or “preloaded,” for subsequentanalysis; (5) providing a common point up to which samples may bepreloaded, whereby timing of loading and injection cycles isstandardized for all samples; and (6) providing enhanced detection andresolution of material regions, e.g., species bands or plugs, within theanalysis channel.

II. Cost Reduction

In general, in fields employing microfabrication, it is desirable toemploy the principles of “shrinking” to optimize the fabricationprocess. Shrinking generally refers to the optimization of a device at afirst scale, followed by the proportional scaling down of the size ofthe device. Shrinking provides a two-fold advantage in device design andmanufacture. First, it provides the readily apparent advantages ofreducing the overall product size. Because of this smaller size, theproduct has smaller space requirements, which can, in turn, be exploitedby integrating the device within smaller overall systems. Further, inmany cases, microfabricated devices, including, e.g., microprocessors,microfluidic devices, and the like, are fabricated from larger wafers ofsubstrate material, e.g., silicon, silica, etc. As such, by reducing thesize of each individual device, one can increase the number of deviceswhich can be produced from a single wafer, reducing the materials costsaccordingly.

Furthermore, this increase in the number of devices produced from asingle wafer also substantially reduces the number of devices that arelost due to flaws in a given wafer or plate. For example, where oneproduces only four devices from a single substrate wafer, a single,small, critical flaw that is wholly contained within one device resultsin a 25% loss, i.e., 1 of 4 devices will include the flaw. However,where one produces 20 different devices from a single wafer, only 5% ofthe devices or 1 of 20 will include the flaw. Thus the cost advantagesof reducing device size are themselves, two-fold.

In the case of the devices of the present invention, dimensions perdevice generally range from about a length and width dimensions of fromabout 5 mm to about 100 mm for a device capable of analyzing multiplesamples, however, larger or smaller devices may also be prepareddepending upon the number of analyses that are to be performed, and thedesired volume of the reagent reservoirs. In preferred aspects, thedevices have length and width dimensions of from about 5 mm to about 50mm.

The optimized channel and well geometries incorporated into the devicesof the present invention allow for a substantially reduced substraterequirement per device. As a result of the reduction in substraterequirements, costs associated with the substrate aspect of the deviceare substantially reduced as a result of an increase in the number ofsubstrates/wafer and decrease in the percentage of substrateslost/wafer. Although described in terms of silica or silicon basedsubstrate materials, it will be readily appreciated that the cost andmaterial savings provided by the present invention are applicable to awide range of substrate materials, e.g., glass, polymeric materials,etc.

III. Increased Throughput

As noted previously, the improved channel and reservoir geometriesincorporated in the devices of the present invention also allow forsubstantially improved throughput for performing particular analyses onmultiple samples. In particular, in any fluidic system, a substantialamount of time is spent simply transporting a material from one locationin the system to another. This is particularly the case in capillaryelectrophoresis systems where transportation of material from onelocation to another location in the system, i.e., from a sample well tothe separation capillary, is carried out electrophoretically. Thisproblem is further magnified where the system is used in the serialanalysis of multiple different samples.

The channel and reservoir geometries incorporated into the devices andsystems of the present invention, on the other hand, result insubstantially shorter transit times from a sample reservoir to theanalysis portion of the device, e.g., channel. The improved geometriesalso permit the incorporation of greater numbers of sample reservoirsper unit area of substrate. Additionally, these geometries permit theperformance of a ‘preloading’ operation which allows the analysis of onesample in the analysis region or channel, while another sample is beingtransported from its reservoir to a location adjacent to the analysisregion or channel. The combination of these elements allows for asubstantial increase in the throughput of the device.

A. Multiple Sample Wells

In one aspect, the devices and systems of the present invention employmultiple sample sources, wells or reservoirs for a given analysischannel, allowing the serial analysis of multiple samples in a singledevice merely by sequentially injecting each of the samples from itsrespective reservoir into the analysis channel, i.e., drawing a samplefrom a first reservoir and injecting it into the analysis channel, thendrawing a sample from a second reservoir and injecting it into theanalysis channel. Although generally described herein in terms of samplewells or reservoirs fabricated into the microfluidic device, it willalso be understood that such sample reservoirs may also exist externallyto the device per se, while remaining in fluid communication with thevarious points on the device as described herein.

Employment of multiple sample reservoirs provides the advantage of beingable to serially analyze multiple samples without having to manuallyload each sample after the analysis of a previous sample has concluded.The devices of the present invention include at least two separatesample reservoirs on a single substrate and in fluid communication witha given analysis channel. Typically, the devices include at least fourseparate sample reservoirs, more typically at least six separate samplereservoirs, preferably, at least eight separate sample reservoirs, andmore preferably, at least twelve separate sample reservoirs, and oftenat least 16 separate sample reservoirs for a given analysis channel.Each of the sample wells is typically in fluid communication with asample loading channel which intersects and is in fluid communicationwith the analysis channel. A load/waste reservoir is typically suppliedin fluid communication with the sample loading channel on the oppositeside of the intersection of the loading channel with the analysischannel. This allows a sample to be loaded by drawing the sample acrossthe intersection and toward the load/waste reservoir. An additionalpreload channel and reservoir is provided in fluid communication withthe sample loading channel on the same side as the samples to be loaded,to permit preloading of one sample while a previous sample is beingtransported along the main channel, e.g., by flowing the sample from itsown well to the load/waste well on the same side of the intersection andthus, not crossing the intersection.

As noted above, the devices of the present invention typically include arelatively high density of sample and other reservoirs per unitsubstrate area. In particular, sample and buffer wells are typicallyincorporated into the device at a density greater than approximately 2reservoirs/cm², preferably, greater than 4 reservoirs/cm², and in somecases, greater than 8 reservoirs/cm². In particularly preferred aspects,the reservoirs included in the devices of the present invention are setat regular spacing. More particularly, such spacing is complementary tospacing found in existing fluid handling systems, e.g., compatible withmultiwell plate dimensions. For example, in preferred aspects, thereservoirs are positioned or arranged in a linear format (e.g., along aline) or gridded fashion at regularly spaced intervals. For example, inpreferred aspects, the wells of the device are arranged on approximately9 mm centers (96-well plate compatible) in a linear or griddedarrangement, more preferably, 4.5 mm centers (384 well plate compatible)and in some cases, on approximately 2.25 mm centers (1536 well platecompatible).

In preferred aspects, the multiple sample reservoirs are disposed atlocations on the substrate on both sides of the analysis channel. Bylocating the sample reservoirs on both sides of the analysis channel,one can minimize the distance, and thus the channel length, between anygiven reservoir and the point on the analysis channel at which thesample is to be injected into that channel, by clustering the samplereservoirs around the point at which the samples are injected into theanalysis channel. By minimizing the length of the channel between thesample reservoirs and the analysis channel, one minimizes the transittime for transporting a sample from its reservoir to the analysischannel. In addition, one also minimizes any effects that result duringthe transportation of the fluids, e.g., adherence of components to thedevice, electrical effects in electroosmotic (E/O), or electrophoreticsystems, which effects may not be desirable prior to injection in theanalysis channel, e.g., electrophoretic biasing or separation of samplecomponents.

In particularly preferred aspects, the sample reservoirs are equallyallocated on both sides of the analysis channel. Thus, in thesepreferred aspects, the device includes at least two, typically at leastthree, preferably at least four, more preferably, at least six, andstill more preferably at least eight separate sample reservoirs on eachside of the analysis channel.

In alternate or additionally preferred aspects, the various samplesources or reservoirs are provided such that the channel distance alongwhich material from each of the sample sources or reservoirs must betransported in order to reach the injection channel (or the sampleinjection channel, as described in greater detail below) issubstantially equal. By “substantially equal” is meant that this channeldistance for any given reservoir is no more than 25% greater or lessthan the same distance for any other sample source or reservoir,preferably, no greater than 15%, more preferably, no greater than 10%,still more preferably, no more than 5% greater or less than the channeldistance for any other sample source or reservoir. In most preferredaspects, these channel distances are within about 2% of each other.

A variety of advantages are gained by providing the sample reservoirsequidistant, in terms of channel distance, from the injection or preloadpoint. Initially, in many applications, fluids such as running buffers,sieving matrices, dynamic coatings, and the like, are added to thechannels of a microfluidic device by depositing these fluids into asingle reservoir, e.g., a buffer or waste reservoir. The fluid thenwicks into the channels of the device by capillary action, and/orhydrodynamic or applied pressure. In these situations, the fluidgenerally travels through equivalently sized channels at approximatelyequivalent rates. Accordingly, where microfluidic devices have variedchannel distances from the injection points, the fluid reaches thereservoirs at the termini of shorter channels first. In the case ofapplied or hydrodynamic pressure, these reservoirs begins filling withthe fluid before the fluid reaches the remaining reservoirs. Thisresults in different fluid levels in the different sample reservoirs,which in turn, results in different dilution levels for each of thesamples, effecting the ability to quantitatively compare samples. Equalchannel distances substantially obviate this problem, as the fluidreaches and fills the various reservoirs at substantially the same timeand rate.

In addition to obviating the dilution problem, incorporation of equalchannel distances, as described herein, also results in sample transittimes, from each of the reservoirs to the injection or preload timebeing equal. This has several advantages. Initially, each sample issubjected to the transit environment for the same amount of time,thereby standardizing any effect such transit time may have. Forexample, in the case of nucleic acids analysis, intercalating dyes areoften mixed with the running buffers within the channels of the device,whereupon the dyes are taken up by nucleic acids traveling through thosechannels, i.e., during transit from a sample source to the analysischannel. Differential transit times can result in differentialincorporation of dye, resulting in different eventual signal intensitiesfrom those different samples. In addition, equal sample loading timesalso provides useful advantages in permitting the operator of the systemto standardize the amount of time required for loading or preloading ofeach sample. In particular, each sample requires the same amount of timeto load or preload, greatly facilitating the operation of themicrofluidic device.

The same advantages obtained by providing sample sources equidistantfrom the injection point are also obtained by providing other variationsin channel structure or geometry whereby the transit time of a givenfluid, e.g., a sample, to or from one sample source is equal to that fora fluid or sample to or from another source. For example, instead ofproviding for sample sources that are equidistant, one can providevariations in the channel width to equilibrate transit time for fluidstraveling to or from two different sources. In particular, samplesources that are nearer to the injection point or preload point, areoptionally provided in fluid communication with the injection or preloadpoint via a wider channel, such that the sample requires substantiallythe same amount of time to travel through that channel from the sourceto the injection or preload point, as a sample source connected by alonger, but narrower channel, under similar or identical materialtransport conditions, e.g., applied electric fields. Similarly, suchchannel variations are useful in equilibrating the amount of timerequired for a fluid to reach any of the sample sources when introducedthrough a common channel, e.g., in filling the device with buffer, etc.,as described in greater detail, herein.

By “substantially the same amount of time,” is meant that the time for asample to travel from its source to the injection point or the preloadpoint on the microfluidic device is within about 25% of the amount oftime for another sample to travel from its source to the injection orpreload point, under the same transport conditions, e.g., appliedpressure or electric fields, i.e., in electrokinetic transport,preferably, no greater than 15%, more preferably, no greater than 10%,still more preferably, no more than 5% greater for any other samplesource or reservoir. In most preferred aspects, the times for samples totravel from different sample sources to the injection or preload pointare within about 2% of each other.

In addition to ensuring that the transit time for each of the multiplesamples is similar to that of other samples within the device, it isalso generally desirable to reduce the amount or length of commonchannel, e.g., loading channel, through which a sample must pass inorder to reach the injection point. In particular, reducing the lengthof the loading channel between its intersection with the sample channelsand the preload/waste channel, reduces the possibility of crosscontaminating materials being present in the ultimate injection. Inparticular, the shortened loading channel is more likely to becompletely flushed before the next sample is actually injected into theinjection cross or intersection. Thus, in at least one aspect of thepresent invention, the intersection of the sample channels with theloading channel is placed closer to the intersection of the load channelwith the load/waste channel, e.g., within about 5 mm, preferably withinabout 4 mm, more preferably within about 2 mm and often within about 1mm, thus creating a loading channel between the sample channelintersection and the injection intersection or preload intersectionhaving a length less than approximately 5 mm, preferably less than about4 mm more preferably less than about 2 mm.

As noted previously, injection of a sample into the main analysischannel typically involves drawing the sample across the intersection ofthe loading channel and the analysis channel. Accordingly, in preferredaspects, the devices and systems of the present invention typicallyincludes a load/waste reservoir and channel in fluid communication withthe sample loading channel on the opposite side of the analysis channelfrom the sample that is to be loaded. Application of a voltage gradientbetween the desired sample reservoir and the load waste/reservoir on theopposite side of the analysis channel then causes material transportthrough the sample loading channel and across the intersection of theloading channel and the analysis channel (also termed the injectionpoint) and into the load/waste channel and reservoir.

Because the devices and systems of the present invention preferablyinclude samples located on each side of the analysis channel, suchdevices also typically include a load/waste reservoir and correspondingchannel on each side of the analysis channel and in fluid communicationwith the sample loading channel. Specifically, the preloading well forsamples on one side of the analysis channel is the load/waste reservoirfor the samples on the other side of the channel. A schematicillustration of this feature is illustrated in FIG. 1.

In brief, FIG. 1A schematically illustrates an intersection between twochannels in a microfluidic device (not shown), e.g., a main analysischannel 100 and a sample loading channel 102. Sample loading channelalso includes first and second sample introduction channels 104 and 106,respectively, in fluid communication with the sample loading channel onopposite sides of the intersection 108, and which sample introductionchannels are also in fluid communication with first and second samplesources 110 and 112, respectively, e.g., a sample reservoir disposed inthe device. In addition to the first and second sample channels, thesample loading channel on each side of the intersection is also in fluidcommunication with first and second load/waste channels 114 and 116,respectively, which are in turn, in fluid communication with first andsecond load/waste reservoirs 118 and 120, respectively.

FIGS. 1B through 1E schematically illustrate the sequential injection ofa sample from each of the first and second sample wells. In particular,FIGS. 1B and 1C show the first sample (indicated by hatching) IIII beingdrawn into the sample loading channel 102 and across the intersection108 of the loading channel with the main channel 100, and into thesecond load/waste channel 116. In electrical material direction systems,e.g., E/O flow or electrophoretic transport systems as generallydescribed herein, this is accomplished by applying a voltage at thefirst sample reservoir 110 and the second load/waste reservoir 120, toachieve material movement along the path of current flow. The plug ofsample material at the intersection is then injected into the mainchannel 100, by applying a voltage at points on the main channel onopposite sides of the intersection 108, i.e., at the buffer and wastereservoirs located at the termini of the main channel 100. Duringinjection, the voltage applied at sample reservoir 110 and secondload/waste reservoir 120 may be removed, e.g., allowing these reservoirsto ‘float.’ However, typically a voltage is maintained at thesereservoirs, so as to achieve a net material flow away from theintersection, e.g., pulling the sample away from the intersection, toavoid any diffusion or leaking of the sample into the intersectionduring analysis.

The second sample (indicated by cross-hatching) is loaded and injectedinto the main channel 104, in the same fashion as the first, as shown inFIGS. 1C and 1D, except that during loading, the voltages are applied atthe second sample reservoir 112 and the first load/waste well 118.

In addition to allowing injection into the analysis channel of samplesfrom both sides of the analysis channel, and in contrast to deviceslacking this feature, incorporation of load/waste reservoirs on bothsides of the analysis channel also permits one sample to be preloadedwhile another sample is being analyzed in the analysis channel, as notedabove.

For example, in typical planar chip based CE devices incorporating aseparation channel and a loading channel in a cross-channel structure, asample is loaded into the separation channel by placing it in areservoir at the terminus of the loading channel and applying a voltageacross the loading channel until the sample has electrophoresed acrossthe intersection of loading channel and the separation channel.Typically, the application of voltage is via an electrode disposedwithin the reservoir or well at the terminus of the given channel (alsotermed a “port”). The plug of material at the intersection is thenelectrophoresed down the separation channel by applying a voltagegradient across the length of the analysis or separation channel. Inorder to avoid disrupting the analysis or separation of the sample,i.e., by interrupting the electric field, one must wait until thatseparation has concluded prior to loading a subsequent sample.

In the channel structures described herein, however, while a firstsample is being analyzed in the analysis channel, e.g., byelectrophoresis, a subsequent sample may be transported to a location inthe loading channel closer or even adjacent to the injection point. Inparticular, by applying an appropriate voltage across the samplereservoir and the load/waste reservoir that is in fluid communicationwith the sample loading channel on the same side of the analysischannel, the sample is transported from its respective reservoir througha portion of the sample loading channel and to that load/wastechannel/reservoir, without crossing the analysis channel. Further, bymaintaining the voltages applied in this preloading procedure at levelssuch that the voltage at the preloading point, e.g., the intersectionbetween load/waste channel 114 and loading channel 102, is substantiallyequal to that at the injection point (108), one can carry out thispreloading without affecting the transportation of material within theanalysis channel, e.g., without producing transverse electric fieldsbetween the loading channel and the analysis channel. Where one isdetermining this voltage at a given intermediate point in a channel(V_(i)), which has a first voltage applied at one end V_(a) and a secondvoltage applied at the other end V_(b), determination of theintermediate voltage is as follows:

$V_{i} = {V_{b} + \frac{R_{b}\;\left( {V_{a} - V_{b}} \right)}{R_{a} + R_{b}}}$Where R_(a) is the resistance between the point at which V_(a) isapplied and the intermediate point at which V_(i) is to be determinedand R_(b) is the resistance between the point at which V_(b) is appliedand the intermediate point where V₁ is to be determined.

Upon completion of analysis of the previous sample, the subsequentsample, already within the sample loading channel, is then merelytransported across the intersection of the loading channel and theanalysis channel, and the plug of sample material at the intersection isthen drawn down the analysis channel as before.

FIGS. 1F through 1I illustrate the same channel intersection structureshown in FIGS 1B through 1E, but wherein that structure is used topreload one sample while a previous sample is being analyzed along themain channel. In particular, FIG. 1F illustrates the post injection ofthe first sample, e.g., as described with reference to FIGS. 1B through1E above. While the first sample is being analyzed, the second sample istransported into a position in the sample loading channel closer to theinjection point, intersection 108, by moving the second sample into thesample loading channel and into the second load/waste channel (see FIG.1G). As shown in FIG. 1H following the completion of the analysis of thefirst sample, e.g., electrophoretic separation, etc., the second sampleis loaded into the analysis channel by drawing it across theintersection 108, and injecting it into the analysis channel (FIG. 1I).This process is then repeated for all the samples that are to beanalyzed.

FIG. 2 schematically illustrates the substantial time savings derivedfrom sample preloading using the devices incorporating this preloadingfeature over devices not incorporating this feature. Briefly, Panel Aschematically illustrates the timing of events required to load andinject multiple samples into the analysis channel using a typicalmicrofluidic device, i.e., which does not include a separate load/wastereservoir on each side of the analysis channel. In particular, loadingany given sample requires the transportation of the sample across theintersection of the analysis channel and the loading channel, followedby the transportation of the sample plug at the intersection down theseparation channel. In these typical devices, no samples are loadedwhile a given sample is being analyzed, as this would result in thedisturbance of the material flow in the analysis channel. Thus, analysisof one sample must be effectively completed prior to loading asubsequent sample, resulting in a sample loading and injection timelinewhere loading and analysis of samples does not overlap, as indicated bythe arrows.

Panel B of FIG. 2 provides a similar timeline for samples seriallyanalyzed in a device of the present invention, which incorporates anadditional load/waste reservoir on the same side as the reservoirscontaining the samples to be loaded. Incorporation of this additionalload/waste reservoir allows the transporting of a sample from itsrespective reservoir into the loading channel, without crossing orotherwise affecting the material flow within the analysis channel, alsotermed “preloading.” As such, while one sample is being transportedalong the analysis channel and analyzed, a subsequent sample may bepreloaded into the loading channel. As shown, the time savings can besubstantial, particularly where multiple samples (e.g., 8, 10, 12, 16 orgreater) are being analyzed.

In order to reduce the amount of dead volume between a preloaded sampleand the analysis channel, it is generally desirable for the load/wastechannel to intersect the sample loading channel at a point that isrelatively close to the intersection of the loading channel and theanalysis channel. In the microfluidic devices of the present invention,the distance between these two intersections is typically less than 5mm, preferably less than 2 mm, more preferably less than 1 mm, andoften, less than 0.5 mm.

In addition, for multiple sample reservoir devices, it is generallydesirable to be able to preload each sample to the same point in thesample loading channel. This permits the standardization andsimplification of timing for preloading, loading and injecting eachsample. In addition, during this preloading time, a myriad of otheroperations may be performed on the sample, including dilution,combination with substrates or other reactants, and the like. As such,it is generally preferable for the load/waste channel to intersect thesample loading channel at a point between all of the sample reservoirsand the main channel. Thus, in preferred aspects, each load wastechannel intersects the sample loading channel at a point between: (1)the intersection of the sample loading channel and the main channel, and(2) the intersection of the sample loading channel with each of thesample channels. Sample loading and preloading in these devices isdescribed in greater detail below.

Finally, in addition to the above described advantages, theincorporation of multiple sample sources, each having a separate path,at least in part, to the injection point, provides at least oneadditional advantage, particularly when that system is applied in CEapplications. Typical CE systems introduce each sample that is beinganalyzed by identical paths, e.g., through the same sample well or viathe same channel or passage. Often times, this can result in anaccumulation within that path of extremely slowly migrating material,e.g., very large nucleic acid molecules or complexes, proteins etc.,which accumulation can result in a general fouling of the separationchannel or capillary.

By including a separate introduction path for each separate sample thatis being analyzed, i.e., as provided herein, as opposed to introducingmultiple samples through the same path, such slow migrating material isgenerally retained within the sample source or the channel that connectsthat source to the common sample loading channel. This effect isparticularly evident in CE applications which include a sieving matrixor medium within the various channels of the device, which matrixaccentuates the differential migration rates of these larger species.

In certain embodiments, the microfluidic devices of the presentinvention optionally include channels that have narrower widthdimensions, particularly at the injection point of the device. Inparticular, by narrowing the dimensions at least at the injectionintersection, one can substantially reduce the size of the sample plugthat is injected into the analysis channel, thereby providing a narrowerband to detect, and thus, greater resolution between adjacent bands.

Providing the analysis channel with narrower width dimensions also isparticularly useful in systems employing laser fluorescent detectionsystems. In particular, by narrowing the width of the injection channeland analysis channel to a range of from about 1× to about 5× of thelaser spot size incident on the detection portion of the analysischannel, one increases resolution of the bands in the analysis channel,as described above, without substantially varying the sensitivity of thedevice. Although less material is being injected into the analysischannel and transported past the detector, the detector is able todetect a greater percentage of that material. Accordingly, in somepreferred aspects, utilizing a laser with a 10 μm spot size, theanalysis channel optionally has a width of from about 10 μm to about 50μm, and preferably from about 20 μm to about 40 μm, and more preferably,about 30 to 35 μm.

IV. Device Description

An example of a device employing improved channel and reservoirgeometries according to the present invention is shown in FIG. 3. Asshown, the device 300 is fabricated from a planar substrate 302, whichhas the various channels fabricated into its surface. A second planarlayer overlays the first and includes holes disposed through it to formthe various reservoirs. This second planar element is then bonded to thefirst.

As shown, the device includes a main separation or analysis channel 304which runs longitudinally down the central portion of the substrate. Themain channel 304 originates in and is in fluid communication with bufferreservoir 306, and terminates, and is in fluid communication with wastereservoirs 308 and 310, via waste channel 312. Sample loading channel314 intersects and is in fluid communication with main channel 304. Asshown, the device also includes multiple separate sample reservoirs 316through 346, inclusive, each of which is in fluid communication withsample loading channel 314, either directly via its respective samplechannels 348–378, or via an intermediate sample channel. Load/wastechannels 380 and 382 are also provided, in fluid communication withsample loading channel 314, on opposite sides of the intersection of thesample loading channel with main channel 304, and between thatintersection and the sample channels on their respective sides of thatintersection. Each of these load/waste channels terminates in one ofload/waste reservoirs 384 and 386, respectively.

The multiple separate sample reservoirs are disposed on both sides ofthe main channel, in order to maximize the number of reservoirs that fiton the substrate, while minimizing the distance that a sample musttravel to reach the analysis channel.

In order to control and direct the electrophoretic movement of materialswithin the device, an electrode is placed into electrical contact witheach of reservoirs 306–310, 316–346, 384 and 386. Again, although thepresent example is described in terms of electrophoretic transport anddirection of materials in the device, it will be readily appreciatedthat other forms of material transport and direction are also envisionedand would be equally benefited by the present invention, e.g.,electroosmotic fluid transport and direction, pressure or pneumaticallydriven fluid transport systems, including those utilizing micropumps, orother displacement driven systems.

In operation, a first sample is disposed within a sample reservoir,e.g., reservoir 316. The sample is transported along sample channel 348to loading channel 314, across the intersection of loading channel 314and main channel 304, by application of an appropriate voltage at samplereservoir 316 and waste reservoir 386. In preferred aspects, appropriatevoltages are also applied at buffer reservoir 306 and waste reservoirs308 and 310, to apply a constraining flow of fluid from the main channelto “pinch” the flow of the sample across the intersection, therebypreventing leakage or diffusion of sample at the intersection. Pinchedloading is described in detail in Published PCT Application No. WO96/04547 to Ramsey et al., which is incorporated herein by reference inits entirety for all purposes.

The sample plug, e.g., the pinched plug, at the intersection of loadingchannel 314 and main channel 304 is then drawn down main channel 304, byapplying a voltage between buffer reservoir 306 and waste reservoirs 308and 310, while reservoirs 316 and 386 are allowed to float. In somecases, appropriate voltages may be applied to these floating reservoirsin order to draw the sample in the loading channel away from theintersection, so as to avoid the leaking of a sample into the analysischannel.

While the first sample is being transported along main channel 304 andbeing subject to the analysis of interest, e.g., electrophoreticseparation, a second sample may be “preloaded” into position in loadingchannel 314 for subsequent analysis. This subsequent sample is preloadedfrom sample reservoir 318 into loading channel 314 and out throughload/waste channel 380 to load/waste reservoir 384, by applying anappropriate voltage at sample reservoir 318 and load/waste reservoir384. As stated previously, the voltages applied at these reservoirs aretypically maintained at levels such that the voltage at the injectionpoint (intersection of channels 304 and 314) is substantially equal tothe voltage at the preload point (intersection of channel 314 and 382),so as to avoid the generation of transverse fields, i.e., a voltagegradient, between the loading channel and the main channel during thepreloading procedure.

Once the first sample has been run down the main analysis channel 304,the preloaded sample in the loading channel 314 is injected across theintersection of the loading channel 314 and the main channel 304 byapplying a voltage across sample reservoir 318 and load/waste reservoir386. The sample plug at the intersection is then transported along mainchannel 304 by again applying an appropriate voltage across the mainchannel, while a third sample is preloaded as described above. This isrepeated for each sample reservoir on each side of the main channel.Thus, as shown, each side of the main channel includes a separate“preloading module” that includes the collection of sample reservoirsand channels, in fluid communication with a sample loading channel. Eachpreloading module includes its own load/waste reservoir and channel influid communication with the sample loading channel, whereby a samplecan be transported from its respective reservoir into the loadingchannel and to a position in the loading channel that is proximal to theintersection of the loading channel and the main channel, withoutaffecting the movement of material in the main channel. As notedpreviously, in order to minimize dead volumes between the preloading ofa sample and the injection of that sample, it is generally preferredthat the load/waste channel for a preloading module, e.g., load/wastechannel 380, intersect its loading channel, e.g., 314, at a point closeto the intersection of the loading channel and the main channel.

A similar channel/reservoir geometry is illustrated in FIG. 4, for adevice which includes 12 separate sample reservoirs, as well as thepreloading features described above. In order to achieve a more compactgeometry, the buffer reservoir 306, waste reservoir 310 and load/wastereservoirs 384 and 386 are located in a row at the bottom of the device.This results in a gridded array of sample/waste/buffer reservoirs,wherein a twelve sample device only occupies approximately one half ofthe substrate area required for the device shown in FIG. 3. Althoughthis device includes fewer sample reservoirs than the device illustratedin FIG. 3 and described above, the sample:area ratio is substantiallyincreased by optimizing the channel and reservoir geometry. Inparticular, where the device shown in FIG. 4 has side dimensions of 17.5mm (e.g., 17.5 mm×17.5 mm), one can obtain 49 separate devices from asingle 5″×5″ square substrate plate or wafer, permitting analysis of 588samples per plate. Assuming the device shown in FIG. 3 is 22.4 mm×37 mm,one can only obtain 15 separate devices or 240 assays per substrateplate.

As noted previously, the devices, systems and methods of the presentinvention are not limited in application to capillary electrophoresisapplications, but may be broadly applied to the field of microfluidics,including fluidic systems employing a variety of material transportmechanisms, including, e.g., electroosmotic transport systems,electrophoretic transport systems and even pressure driven systems.However, where these devices, systems and methods are used in capillaryelectrophoresis applications, i.e., to separate sample components, e.g.,nucleic acid fragments, it is generally desirable to reduce the level ofelectroosmotic flow within the channels of the device, therebyoptimizing the differential mobility of differentially charged or sizedspecies within the system, and thus their separability.

Accordingly, in preferred aspects, where the devices and systems of theinvention are employed in capillary electrophoresis applications, thechannels of the device are pretreated with a dynamic sieving matrix.Such dynamic sieving matrices typically comprise charged polymers, e.g.,linear polyacrylamide polymers, which are capable of binding the wallsof the capillary channels, thereby shielding the charged surfaces ofthese walls, and reducing electroosmotic flow. Examples of particularlypreferred dynamic sieving matrices include those discussed in U.S. Pat.No. 5,264,101, incorporated herein by reference in its entirety for allpurposes, as well as the GeneScan™ sieving buffers available from PerkinElmer Corp.

A device incorporating an alternate channel geometry according to thepresent invention is illustrated in FIG. 5. As shown the device includesa channel geometry that is similar to that shown in FIG. 4. Inparticular, as shown, the microfluidic device 500 fabricated fromsubstrate 502 includes main channel 504 that has disposed at its terminibuffer reservoir 506 and waste reservoir 508. Main channel 504 isintersected by and in fluid communication with first ends of sampleloading channels 512 and 514 (on the left and right sides respectively,as shown). Sample channel 512 is in fluid communication at its secondend with sample reservoirs 516–526, via sample channels 542–550,respectively, whereas sample loading channel 514 is in fluidcommunication at its second end with sample reservoirs 528–538 viasample channels 552–562, respectively. Sample preload waste channels 582and 584 intersect sample loading channels 512 and 514, respectively, atpoints proximal to the injection point or intersection.

The device illustrated performs substantially the same operations as thedevices shown in FIGS. 3 and 4. However, as shown, the device 500includes sample channels 540–562 that are of substantially equallengths, from their respective reservoirs to the point at which theyintersect their respective sample loading channel (512 or 514). Thedevice shown provides all of the advantages delineated above.

Further, in an alternate aspect as described above, and with referenceto FIG. 5, the intersection of sample loading channels 512 and 514 withtheir respective sample/preload channels 582 and 584, are placed closerto the intersection of these channels with their respective samplechannels, e.g., 540–550 and 552–562, respectively, in order to minimizethe length of the sample loading channels and thereby reduce thepossibility of cross-contamination of samples during preloading. Inpreferred aspects, this loading channel length between theseintersections is less than about 5 mm, and preferably, less than about 2mm.

As noted above, the devices and systems described herein may generallybe used in the analysis of chemical and biochemical materials. Forexample, in at least one aspect, the present invention provides for theuse of such devices and systems in separating component elements of thesample materials, e.g., nucleic acids, proteins, or other macromolecularspecies, as well as differentially charged materials. The devices of thepresent invention are optionally provided in kits. The kits of theinvention optionally comprise one or more of the following: (1) anapparatus or apparatus component as described herein, e.g., themicrofluidic device(s) described above); (2) instructions for practicingthe methods described herein, and/or for operating the apparatus orapparatus components herein; (3) one or more assay component(s), e.g.,reagents, fluorescent dyes, standards, sieving matrices or the like; (4)a container for holding apparatus or assay components, and, (5)packaging materials.

The above-described microfluidic devices are typically placed in anelectrical controller unit that disposes an electrode in each of thereservoirs of the device to carry out the operations of the device, asdescribed herein. The controller unit delivers appropriate currentsthrough the electrodes that are in contact with the reservoirs of thedevice, in order to direct material through the channels of the device.The currents delivered by the controller are typically current and timeprofiles for each electrode that are entered into a computer by a user,which computer is operably connected to the controller. The computerthen instructs the controller in the application of currents to thevarious electrodes to move material through the channels of the devicein a controlled manner, e.g., providing sufficient current levels toachieve controlled electrokinetic material transport, as describedabove.

The invention is further described with reference to the followingnonlimiting examples.

EXAMPLES Example 1 Multisample Analysis

A 16 sample capacity device, or LabChip™, having the geometry shown inFIG. 3, was fabricated from a 100 mm diameter white crown glass waferhaving a thickness of 500 μm. A wafer was used for its compatibilitywith most commercially available photolithography equipment. Channels 75μm wide and 12 μm deep, and having the configuration shown, were etchedin the glass substrate using standard photolithographic techniques.Holes were drilled through a separate piece of glass 5 inches on a side,whereby the holes corresponded to the termini of the various channels.The two pieces of glass were thermally bonded to form the channel andwell structure shown. The device having dimensions of 22.4 mm×37 mm wascut from the larger material.

Sieving buffer was prepared by weighing 2.5 grams GeneScan Polymer(Perkin Elmer Corp.), 0.5 g of Genetic Analysis Buffer (Perkin ElmerCorp.) and 2.5 ml water into a 20 ml scintillation vial, which was thenvortexed for 30 seconds. One μl of Syber Green 1 DNA intercalation dye(Molecular Probes Inc.) was added to 0.5 ml of the sieving buffer whichwas again vortexed for 30 seconds in a 1.5 ml Eppendorf tube. Five μlPCR Marker (Promega Corp.) containing 6 DNA fragments ranging in sizefrom 50 to 1000 bp was mixed with 15 μl of the buffer containing theSyber Green and vortexed.

The channels in the LabChip™ were filled with 3.5% GeneScan™ buffer(Perkin-Elmer Corp.) by applying 5 μl to the buffer well and thenapplying slight pressure with a syringe for 5 seconds on the well. Thisbuffer contains a polymer which retards the migration of DNA relative toits size and also modifies the walls of the channel to reduceelectroosmotic flow. Four μl of the GeneScan buffer was then added tothe buffer and waste wells.

A DNA standard, PhiX174 cleaved with HinfI (Promega Corp.), was diluted50:1 in 3.5% GeneScan™ buffer containing 1 μM SyberGreen DNAintercalating dye (Molecular Probes, Inc.) and 4 μl of this solution wasadded to each of the 16 sample wells. The device was then placed under aNikon inverted Microscope Diaphot 200, with a PTI Model 814 PMTdetection system, for epifluorescent detection. An Opti-Quip 1200–150050 W tungsten/halogen lamp coupled through a 40× microscope objectiveprovided the light source. Excitation and emission wavelengths wereselected with a FITC filter cube (Chroma, Brattleboro Vt.) fitted withappropriate filters/dichroic mirrors. Reagent well currents and voltageson the chip were controlled using a voltage controller having a separatecontrollable electrode for each of the separate reservoirs on themicrofluidic device. The serial injection of samples proceeded along thefollowing cycle:

-   -   Step 1: Initial Sample Preload (45 secs.)    -   Step 2: Sample Load (5 secs.)    -   Step 3: Inject (1 sec.)    -   Step 4: Pull Back (2 secs.)    -   Step 5: Run/Next Sample preload (85 secs.)    -   Step 6: Next Sample Load (5 secs.)    -   Step 7: Repeat Steps 3–6

An example of the cycle of currents applied at the various reservoirsduring a single cycle is provided in the following table. The samplepull back step was inserted to pull the sample away from theintersection of the loading channel with the main channel, and thusprevent bleeding over of the sample. Also, during the loading steps,e.g., steps 2 and 6, a pinching flow was delivered to the intersectionsuch that the flow of sample would not diffuse into the main channel asa result of convective effects. Applied voltages were controlled using acurrent based control system, e.g., as described in commonly assignedU.S. patent application Ser. No. 08/678,436, filed Jul. 3, 1996, andincorporated herein by reference in its entirety for all purposes.Currents applied for each of the above steps were as shown in Table 1,below. The voltage applied to main buffer reservoir 306 was controlledat a level at which it provided an appropriate balancing current in theremainder of the system:

TABLE 1 Sample Load/Waste Waste Sample Current Load/Waste Current BufferCurrent Step Well (μA) Well (μA) Wel (μA) 1 332 −7 386 10 310 −2 2 332−7 384 10 310 −2 3 332 5 384 5 308 −12 4 332 1 384 1 308 −8 5 334 −7 38610 308 −7.5 6 334 −7 384 10 310 −2

The results of the first separation using this method are shown in FIG.6. As is clear from this figure, this method of performing capillaryelectrophoresis yields high resolution in a substantially reducedtime-frame. Further, no degradation of resolution was seen throughseparation of all 16 samples.

Example 2 Determination of Cross Contamination Levels for SuccessiveSamples

In order to ascertain whether successive runs in the device experiencedany cross contamination of samples, two different nucleic acid fragmentsamples and a plain buffer sample were run in succession and examinedfor contaminating effects.

Each well of the 16 well device described above, was loaded with eitherthe PCR Marker, the PhiX174 cleaved with HAEIII or plain buffer. Thewells were loaded such that they would be injected successively in thisorder. The fluorescence data for each run was plotted as a function oftime.

FIGS. 7A, 7B and 7C show plots of successive injections of PCR Marker,PhiX174/HaeIII and buffer blanks. FIG. 7B illustrates that no spuriousfluorescence peaks are detectable bleeding over from the previous PCRMarker run, into the PhiX174/HaeIII run. Further, FIG. 7C shows thateven in a plain buffer run, there are no detectable levels of crosscontamination from the prior DNA containing samples.

Example 3 Narrow Channel Injection

A microfluidic device incorporating the channel geometry shown in FIG.5, was prepared as described in Example 1, above, except that the widthof all channels of the device was reduced to 30 μm, while the channeldepth was maintained at approximately 12 μm. This was used to comparewith a microfluidic device having the geometry shown in FIG. 4, buthaving channel widths of approximately 70 μm, as described in Example 1.The length of the separation channels in the two devices wassubstantially equivalent.

The two devices were prepared with sieving buffer, as described above,and each was used to separate a nucleic acid standard 100 base pairladder, commercially available from Promega Corp., Madison Wis. Theresults of the separations in the narrow channel and wide channel deviceare illustrated in FIGS. 8A and 8B, respectively. As is readilyapparent, the resolution obtained in the narrow channel device (FIG. 8A)is substantially enhanced over the device incorporating wider channels(FIG. 8B).

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

1. A microfluidic device comprising: a body structure comprising first and second microchannels, the microchannels intersecting at a first intersection, the body structure further comprising a plurality of reservoirs, the plurality of reservoirs comprising first, second, third, and fourth sample reservoirs and first, second, and third waste reservoirs, the plurality of reservoirs comprising a total of at least 8 reservoirs disposed on a first surface of the body structure, wherein each sample reservoir is fluidly connected to the first intersection by a substantially equal fluid path, wherein each sample reservoir is fluidly connected to the first waste reservoirs through a fluid path comprising the first microchannel, and wherein each sample reservoir is fluidly connected to one of the second or the third waste reservoirs through only on fluid path, wherein said one fluid path does not comprise the first microchannel; and a detector that detects one or more analytes in the first microchannel.
 2. The microfluidic device of claim 1, wherein the plurality of reservoirs comprises a total of at least 16 reservoirs on a first surface of the body structure.
 3. The microfluidic device of claim 1, wherein the first sample reservoir is disposed on a first side of the second microchannel and the second waste reservoir is disposed on a second side of the second microchannel, which second microchannel intersects the first microchannel at the first intersection.
 4. The microfluidic device of claim 3, wherein the first sample reservoir is connected to the second microchannel by a fluid path comprising a third microchannel, which third microchannel intersects the second microchannel at a second intersection upstream of the first intersection.
 5. The microfluidic device of claim 4, wherein the second sample reservoir is disposed on a first side of a fourth microchannel and the third waste reservoir is disposed on a second side of the fourth microchannel, which fourth microchannel intersects the first microchannel at the first intersection.
 6. The microfluidic device of claim 5, wherein the second sample reservoir is connected to the fourth microchannel by a fluid path comprising a fifth microchannel, which fifth microchannel intersects the fourth microchannel at a third intersection.
 7. The microfluidic device of claim 6, wherein the detector also detects one or more analytes in one or more of the second microchannel, the third microchannel, the fourth microchannel or the fifth microchannel.
 8. The microfluidic device of claim 7, wherein the detector detects an analyte in the first, third and fifth microchannels.
 9. The microfluidic device of claim 1 or 2, further comprising one or more controller for flowing material in the microfluidic device, wherein the one or more controller includes one or more of: a voltage regulator; a pressure regulator; or a hydrodynamic force regulator.
 10. The microfluidic device of claim 3, wherein the third sample reservoir is disposed on the first side of the second microchannel.
 11. The microfluidic device of claim 3, wherein the first and second microchannels of the device are approximately the same width and depth.
 12. The microfluidic device of claim 3, wherein the first and second microchannels of the device comprise at least one dimension between about 1 and 500 microns.
 13. The microfluidic device of claim 3, wherein the first and second microchannels of the device have a depth of about 12 microns and a width of between about 30 and 70 microns.
 14. The microfluidic device of claim 4, wherein the second intersection is proximal to the first intersection.
 15. The microfluidic device of claim 14, wherein the second intersection is within about 5 mm of the first intersection.
 16. The microfluidic device of claim 14, wherein the second intersection is within about 4 mm of the first intersection.
 17. The microfluidic device of claim 14, wherein the second intersection is within about 2 mm of the first intersection.
 18. The microfluidic device of claim 14, wherein the second intersection is within about 1 mm of the first intersection.
 19. The microfluidic device of claim 1, wherein the first microchannel is treated to reduce electroosmotic flow in at least a portion of the first microchannel.
 20. The microfluidic device of claim 1, the body structure having an interior portion and an exterior portion, wherein the first microchannel is disposed in the interior portion.
 21. The microfluidic device of claim 1, wherein the first and the second sample reservoirs are disposed on opposite sides of the first microchannel.
 22. The microfluidic device of claim 1, the plurality of reservoirs comprising at least four separate sample reservoirs on each side of the first microchannel.
 23. The microfluidic device of claim 1, the plurality of reservoirs comprising at least six separate sample reservoirs on each side of the first microchannel.
 24. The microfluidic device of claim 1, the plurality of reservoirs comprising at least eight separate sample reservoirs on each side of the first microchannel.
 25. The microfluidic device of claim 1, wherein the fluid paths between each of the sample reservoirs and the first intersection are within about 25% of the same length.
 26. The microfluidic device of claim 1, wherein the fluid paths between each of the sample reservoirs and the first intersection are within about 15% of the same length.
 27. The microfluidic device of claim 1, wherein the fluid paths between each of the sample reservoirs and the first intersection are within about 10% of the same length.
 28. The microfluidic device of claim 1, wherein the fluid paths between each of the sample reservoirs and the first intersection are within about 5% of the same length.
 29. The microfluidic device of claim 1, wherein the fluid paths between each of the sample reservoirs and the first intersection are within about 2% of the same length.
 30. The microfluidic device of claim 1, further comprising a material transport system.
 31. The microfluidic device of claim 1, further comprising at least one electrode in contact with the first waste reservoir.
 32. The microfluidic device of claim 1, wherein the first microchannel comprises a detection window.
 33. The microfluidic device of claim 1, wherein the first microchannel comprises a separation medium disposed therein.
 34. The microfluidic device of claim 1, wherein the first microchannel comprises a sieving matrix disposed therein.
 35. The microfluidic device of claim 1, wherein the plurality of reservoirs are arranged at a density of greater than about 2 reservoirs/cm².
 36. The microfluidic device of claim 1, wherein the plurality of reservoirs are arranged at a density of greater than about 4 reservoirs/cm².
 37. The microfluidic device of claim 1, wherein the plurality of reservoirs are arranged at a density of greater than about 8 reservoirs/cm².
 38. The microfluidic device of claim 1, wherein the plurality of reservoirs are positioned or arranged in a linear or gridded format.
 39. The microfluidic device of claim 38, wherein the plurality of reservoirs are arranged at regularly spaced intervals.
 40. The microfluidic device of claim 38, wherein the plurality of reservoirs are arranged on one or more of: 9 mm centers; 4.5 mm centers; or 2.25 mm centers.
 41. The microfluidic device of claim 1, wherein the body structure comprises a planar substrate, wherein the first surface is rectangular, and wherein a length or width of the first surface is between about 5 mm and about 100 mm.
 42. The microfluidic device of claim 41, wherein the length or width of the first surface is between about 5 mm and about 50 mm.
 43. The microfluidic device of claim 1, wherein the body structure comprises one or more material selected from: a silica-based substrates, glass, quartz, silicon, polysilicon, gallium arsenide, polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene polysulfone, polycarbonate, polymethylpentene, polypropylene, polyethylene, polyvinylidine fluoride, and ABS (acrylonitril-butadiene-styrene copolymer). 